Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.     

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Peptide-containing neurons in explant cultures of guinea-pig myenteric plexus during development in vitro: Gross morphology and growth patterns

Summary The gross morphology and growth patterns of substance P, enkephalin-, somatostatin and vasoactive intestinal peptide-immunoreactive neurons have been studied in explant cultures of the myenteric plexus taken from beneath the newborn guinea-pig taenia coli, grown for up to 4 weeks in vitro. Substance P and enkephalin-immuno-reactive neurons were more abundant than somatostatin and vasoactive intestinal peptide-immunoreactive neurons. The peptide-containing neuronal cell bodies were clearly visible in culture and exhibited characteristic gross morphologies similar to those described in situ, although some overlap of shape between populations containing different peptides was seen. All four types of peptide-containing fibres were found in the outgrowth and central areas of the cultures. In the case of substance P and somatostatin, the density and pattern of labelling in the central, neuronal area of the cultures resembled that previously seen in the myenteric plexus of the newborn guinea-pig caecum in situ, while the density of the enkephalin-immunoreactive fibres was greater, and that of the vasoactive intestinal peptide-immunoreactive fibres less than that seen in situ. These observations suggest that subpopulations of myenteric neurons containing different peptides may be differentially affected by the culture environment. Possible contributory factors are discussed.     

Isolation of Neuropeptide-Containing Vesicles from the Guinea Pig Ileum

Three distinct vesicle fractions enriched 40-60 times in the neuropeptides substance P, somatostatin, and vasoactive intestinal peptide (VIP) were prepared from the myenteric plexus of guinea pig ileum by density gradient centrifugation in a small zonal rotor. Mean densities (in g X ml-1) and diameters (in nm) of the three classes of vesicles were: substance P, 1.123, 65; somatostatin, 1.138, 37; VIP, 1.148, 110; standard deviations were about 5%. These peaks were distinct from the peak of acetylcholine-containing vesicles of density 1.066 g X ml-1 and diameter 61 nm. When a relatively mild method of homogenization was used a second peak of acetylcholine appeared in the same region of the gradient as VIP and the VIP was larger. This may represent a class of vesicles containing both acetylcholine and VIP, though cosedimentation of two classes of vesicles of almost the same density and similar fragility, one containing VIP and the other acetylcholine, cannot be entirely excluded on present evidence.     

Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation

Biochemical, histochemical and neurophysiological data suggest that substance P and somatostatin are neurotransmitters for primary afferent neurons. This study used intrathecal administration of these peptides and others (neurotensin and vasoactive intestinal polypeptide) in chronically catheterized, environmentally adapted, freely moving rats to evaluate their effects on unconditioned behavior. Substance P and somatostatin each elicited behaviors which were dose related. The behaviors included caudally directed biting and licking along with hindlimb scratching, writhing and retching. The behavioral responses were rapid in onset (1 min) and, in the case of substance P, short in duration (3 min). Vehicle, neurotensin and vasoactive intestinal polypeptide were without effect. These results demonstrate the ability of substance P and somatostatin to induce behavior in rats upon intrathecal administration and extend previous studies in mice.     

Selective localization of vasoactive intestinal peptide and substance P in human eosinophils

Extracts of purified human eosinophils had a mean concentration of 72 fmol of immunoreactive vasoactive intestinal peptide and 21 fmol of substance P per 10(7) eosinophils, that were significantly higher than the content of immunoreactivity of the same neuropeptides in neutrophils, mononuclear leukocytes, and platelets. In contrast, the lower concentrations of calcitonin gene-related peptide and somatostatin were similar in extracts of all leukocytes. Chromatography of the peptides from eosinophils confirmed their identity with vasoactive intestinal peptide and substance P from neuroendocrine sources. Stores of some neuropeptides may endow eosinophils with unique roles in host defense and hypersensitivity reactions.     

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in pelvic nerves supplying the posterior large intestine of the toad,Bufo marinus

The distribution and colocalization of neuropeptides and 5-hydroxytryptamine in the posterior portion of the large intestine of the toad was studied using single- and dual-label immunohistochemistry. Neurons containing colocalized galanin/somatostatin or vasoactive intestinal peptide alone were observed along intramural pelvic nerves. Some of the galanin/somatostatin neurons also contained 5-hydroxytryptamine. Synaptic boutons containing colocalized calcitonin gene-related peptide/vasoactive intestinal peptide were associated with the galanin/somatostatin neurons. The muscle of the large intestine was also innervated by axons containing galamin/somatostatin, vasoactive intestinal peptide/calcitonin gene-related peptide or vasoactive intestinal peptide alone. Nerve fibres containing calcitonin gene-related peptide/substance P, probably representing primary afferent nerves, were also associated with muscle bundles. Submucosal blood vessels carried dense plexuses of fibres containing vasoactive intestinal peptide alone or and calcitonin gene-related peptide/substance P. Adrenergic perivascular nerves also contained galanin and neuropeptide Y.     

Recombinant Cholinergic Differentiation Factor (Leukemia Inhibitory Factor) Regulates Sympathetic Neuron Phenotype by Alterations in the Size and Amounts of Neuropeptide mRNAs

The cholinergic differentiation factor (CDF) in heart cells is identical to leukemia inhibitory factor (LIF). Recombinant CDF/LIF was shown to alter dramatically neurotransmitter production as well as the levels of several neuropeptides in cultured rat sympathetic neurons. Here it is shown that these changes are likely to be caused by alterations in the mRNA for these proteins and peptides. Growth in 1 nM recombinant CDF/LIF induces mRNA for acetyl CoA: choline-O-acetyltransferase [EC 2.3.1.6; choline acetyltransferase (ChAT)], somatostatin (SOM), substance P, and vasoactive intestinal polypeptide while lowering mRNA levels of tyrosine hydroxylase (EC 1.14.16.2) and neuropeptide Y (NPY). In addition, the sizes of the mRNAs for ChAT, SOM, and NPY are larger after recombinant CDF/LIF treatment.     

The endocrine pancreas of Alligator mississippiensis

Summary Immunocytochemical methods for light and electron microscopy were used to demonstrate the regulatory peptides present in the endocrine pancreas of thealligator, Alligator mississippiensis.The peptides studied included insulin, glucagon (pancreatic and enteric), somatostatin, pancreatic polypeptide (avian, bovine and human), vasoactive intestinal polypeptide, substance P, metenkephalin, -endorphin, C-terminal gastrin/CCK and gastric inhibitory polypeptide. Endocrine cells were detected using antisera to insulin, pancreatic glucagon, somatostatin and avian pancreatic polypeptide, whereas peptidergic nerves were stained with antisera to vasoactive intestinal polypeptide. All other antisera were unreactive in the alligator pancreas. The peptide-containing structures were identified ultrastructurally by both the semithin/thin and immuno-gold methods. The results showed that five of the regulatory peptides commonly detected in the mammalian pancreas were immunologically recognisable in the alligator. In addition, the ultrastructural appearance of the peptide-containing cells was clearly distinct from that reported in mammals.     

The distribution of neuropeptides in the ocular tissues of several mammals: A comparative study

1. The distribution of several neuropeptides (vasoactive intestinal peptide, substance P, somatostatin and neurotensin) was assessed in ocular tissues from the cow, sheep, rabbit and rat.2. Vasoactive intestinal peptide was most abundant in the choroid and sciera in all species except the rat. Substance P was most abundant in the retina of cow and rat and in the iris/ciliary body of sheep and rabbit. Somatostatin and neurotensin were most abundant in the retina of all species examined.3. Regulatory peptides thus display distinct regional distributions within the ocular tissues of a single species of mammal and, in addition, exhibit interspecific variation.     

Immunohistochemical characteristics of nerve fibres supplying the porcine vas deferens. A colocalisation study

 Double-labelling immunofluorescence was used to investigate the coexistence of the catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase and several neuropeptides including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P in nerve fibres supplying the vas deferens in juvenile and adult pigs. The study has revealed three major populations of nerve terminals innervating the organ: (1) noradrenergic fibres; (2) non-noradrenergic (putative cholinergic) fibres containing vasoactive intestinal polypeptide, neuropeptide Y and somatostatin, supplying almost exclusively the lamina propria; and (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. The population of noradrenergic nerves can be divided into three subpopulations: a somatostatin-containing, a Leu⁵-enkephalin-containing and a subpopulation immunonegative to the peptides investigated, in descending order of magnitude. Coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non-noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the porcine vas deferens and its blood vessels is discussed. Accepted: 23 October 1996     

Imagerie phénotypique et peptides radiomarqués au gallium-68 : au-delà des analogues de la somatostatine

Receptor targeting with radiolabeled peptides has become very important in nuclear oncology in the past few years. The most frequently used peptides in the clinic are analogs of somatostatin. However, other radiolabeled analogs have also been developed and assessed in vitro and in vivo and some of them are already in clinical use. For instance, radiolabeled analogs of alpha-melanocyte-stimulating hormone (alpha-MSH), neurotensin, vasoactive intestinal peptide (VIP), bombesin (BN), substance P (SP), cholecystokinin (CCK), integrins… This review focuses on gallium-68 radiolabeled peptides developed for PET imaging, except for somatostatin analogs, which are discussed in another article.     

Studies on hormonal regulation of lipolysis and lipogenesis in fat cells of various mammalian species

1. Corticotropin-stimulated lipolysis in adipocytes of rats, mice, hamsters, guinea pigs and rabbits. Melanotropins elicited high lipolytic activity only in guinea pig and rabbit adipocytes. Opiate peptides were active only in rabbit adipocytes. Pituitary and chorionic gonadotropins and somatotropin were lipolytic in guinea pig adipocytes. Other hormones tested including prolactin, somatostatin, substance P, neurotensin, angiotensin II, thyrotropin releasing hormone and pancreatic polypeptide were devoid of lipolytic activity in all of the adipocytes studied. 2. In the rabbit adipocytes gamma-melanotropin was lipolytic only at high doses. At these doses the peptide inhibited the lipolytic response to a high dose of corticotropin. 3. Lipolysis stimulated by vasoactive intestinal peptide and epinephrine in rat adipocytes was antagonized by insulin. The lipolytic hormones corticotropin, epinephrine, vasoactive intestinal peptide and secretin suppressed basal and insulin-stimulated lipogenesis.     

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig

Summary The peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.     

An immunocytochemical study of endocrine pancreas of snakes

Summary The pancreas from eleven species of snakes representing both advanced and primitive families has been investigated for the presence of eleven regulatory peptides reported to occur in the mammalian endocrine pancreas. Of the eleven peptides studied, insulin, pancreatic glucagon and somatostatin were present in endocrine cells within the islets of all the species investigated. The neuropeptide, vasoactive intestinal polypeptide, was located within nerve terminals innervating the islets in the Boidinae, Colubrinae, Elaphidae and Crotalidae but absent from the Natricinae investigated.No immunoreactivity was demonstrable with the antisera to substance P, met-enkephalin, C-terminal gastrin, bombesin, glicentin and gastric inhibitory polypeptide. Pancreatic polypeptide-like immunoreactivity was demonstrable only in the boid snakes and exclusively stained by a C-terminal specific antiserum.     

Neuropeptides and septo-hippocampal neurons: Electrophysiological effects and distributions of immunoreactivity

The septo-hippocampal neurons (SHNs), located in the medial septum, project to the hippocampal formation. The population of SHNs, as shown by single unit recordings in urethane-anesthetized rats, is heterogeneous, both in terms of patterns of spontaneous activity (a significant proportion of the SHNs display a characteristic rhythmically bursting activity at about 4 Hz) and of conduction velocity. Their average rate of spontaneous discharge is quite high (20 impulses per second). They are excited by the iontophoretic application of acetylcholine and various cholinergic agonists. They are also excited by some peptides such as substance P and TRH. Parallel studies in aged animals show that the physiological properties of the SHNs are altered, while their pharmacological properties seem to be unchanged. Immunohistochemical investigations using antibodies against various peptides and a monoclonal antibody against choline acetyltransferase (ChAT) show that SHNs retrogradely-labeled from the hippocampus often contain ChAT, less frequently galanin-like immunoreactivity and in a few cases enkephalin, luteinizing hormone-releasing hormone, or calcitonin gene-related peptide. In contrast, cholecystokinin, vasoactive intestinal peptide, substance P, somatostatin, dynorphin-B and neurotensin, although present in some medial septal neurons, were never observed in neurons projecting to the hippocampus.     

ACTH release in pituitary cell cultures. Effect of neurogenic peptides and neurotransmitter substances on ACTH release induced by hypothalamic corticotropin releasing factor (CRF).

The effects of various neurogenic peptides and neurotransmitter substances on the release of ACTH induced by hypothalamic corticotropin releasing factor (HY-CRF) were investigated using monolayer cultured anterior pituitary cells. Test substances were given in combination with 0.05-0.1 hypothalamic extract (HE)/ml, because HE evoked a significant ACTH release and a linear dose response relationship was demonstrated sequentially between 0.0165 HE/ml and 0.5 HE/ml. Relative high doses of lysine-vasopressin showed a slight additive effect on the release of ACTH induced by 0.1 HE/ml. Leu-enkephalin, dopamine, prostaglandin E1 and E2 slightly reduced the release of ACTH induced by HY-CRF, but the inhibitory effect of these substances were not dose-related. Other tested substances including luteinizing hormone releasing hormone, thyrotropin releasing hormone, somatostatin, melanocyte stimulating hormone release inhibiting factor, beta-endorphin, neurotensin, substance P, vasoactive intestinal polypeptide, angiotensin II, norepinephrine, serotonin, acetylcholine, histamine and gamma-amino butyric acid showed neither agonistic nor antagonistic effect on the release of ACTH induced by HY-CRF. These results indicate that the release of ACTH is controlled specifically by HY-CRF and corticosterone, and modified slightly by some other substances such as vasopressin and prostaglandins, and that the effect of most other neurogenic peptides and neurotransmitter substances is negligible or non-physiological at the pituitary level.     

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.     

Peptides and neurotransmission in the central nervous system

Radioimmunoassays of brain extracts have shown that several peptides occur in high concentrations in the CNS. The releasing-factor peptides TRF, LRF, somatostatin, CRF and GRF have the highest concentration in the hypothalamic extracts. High levels of somatostatin, CCK octapeptide, neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are found in cortical extracts. Substance P, CCK, NPY, and enkephalins are present in high concentrations in basal ganglia and mesolimbic areas. Pharmacological doses of these peptides result in several behavioural and vegetative effects. Immunocytochemical studies show that the CNS peptides are localised in neurones and in synaptic vesicles. In vitro studies with brain tissues show that peptides are capable of modifying the ongoing classical neurotransmission. In depressive patients several neuropeptides (CCK, CRF and NPY) have been shown to have low CSF levels. Patients dying of senile dementia have low cortical levels of somatostatin, CRF and substance P. In schizophrenic patients CCK peptides have shown to improve some symptoms. At present the therapeutic potentials of peptides are poorly known. More studies are required to understand their role in neurotransmission and related pathological states.     

Effect of gut regulatory peptides on intestinal luminal fluid in the rat

The effect of intravenous gastrointestinal peptide hormone administration on net fluid transport in the small intestine was assessed in the rat. An increased fluid content was observed during vasoactive intestinal peptide, gastric] inhibitory peptide, and neurotensin infusions, and a decreased content with somatostatin, substance P and pancreatic polypeptide, by comparison with the control series. Motilin had no significant effect on luminal fluid volume. These results suggest that several of the gastrointestinal regulatory peptides may have an influence on the processes of fluid absorption and secretion by the small intestine.